Protein preparation for the prevention and therapy of periodontitis

ABSTRACT

The present invention relates to a topical protein preparation to be used for the therapy of periodontitis and diseases of the teeth and of the tissues of the oral cavity, comprising a mixture of transferrin with at least one protein selected from albumins, lysozymes, protease inhibitors, and mixtures thereof.

TECHNICAL FIELD

The present invention relates to a protein preparation for preventingand curing bacterial pathologies and disorders of the oral cavity, inparticular periodontitis and dental caries.

BACKGROUND ART

Periodontal disease comprises a group of disorders that affect thesupporting structures of the teeth, leading to their progressivepathologic-anatomical deterioration and ultimately to a possiblecomplete loss of the dental elements. From an epidemiological point ofview, it has been stressed that periodontal disease, in its variousforms, affects a considerable number of individuals (30-40% between 25and 40 years of age, and 60-70% of individuals over 40).

From a clinical point of view, the disease is characterized by guminflammation, edema, provoked or spontaneous bleeding, apical migrationof the connective attachment, with the forming of periodontal pockets,bone reabsorption, marked dental mobility and, in some cases, gumrecession.

A few years ago it was demonstrated that periodontitis, in all of itsclinical forms, has a bacterial etiology and that the microorganismsthat most frequently cause this disease are Gram-negative obligateanaerobes such as Porphyromonas gingivalis.

Dental caries is instead a demineralizing disorder that affects teeth atall levels and can cause extensive crown mutilations, bacterialdisorders of the periapical tissues or even loss of the affected dentalelements. Approximately 50% of adult individuals have at least fourcaries-related lesions treated and to be treated, and approximately 30%of adult individuals have over 50% of their teeth affected by caries.

Clinically, the disease is characterized by demineralization of thedental enamel and of the dentin in various stages of progress, until itaffects the pulp space. When the lesion passes beyond the enamel-dentinborder, a phlogistic reaction of the pulp tissues is constantlyobserved, with the formation of reaction dentin in some cases.

It has been demonstrated that caries is produced by an acid attack onthe dental surfaces, linked to the fermentative metabolism of sugars inthe diet on the part of some bacterial species capable of adhering tothe dental enamel. The species most frequently responsible for caries isStreptococcus mutans.

It has been extensively demonstrated that the ability of microorganismsto produce infections is linked to the possibility of their adheringspecifically or non-specifically to the different tissues, which allowsthe microorganisms to subsequently colonize the tissues extensively. Inthe pathogenesis of the most common infections of the oral cavity,bacterial adhesion and subsequent colonization of dental surfaces andsoft tissues is undoubtedly of primary importance. The dental surface isnormally covered by a thin film (acquired film) formed by the salivaryglycoproteins that adsorb selectively on the surface of the tooth,allowing colonization on the part of specific microorganisms andpreventing it to others. Furthermore, saliva is constituted by a seriesof protein-based substances having an antibacterial activity.Consequently, by possessing an antibacterial activity and ananti-adhesive activity of a competitive type against pathogenic species,saliva has a very important role in the qualitative and quantitativecontrol of the microbial flora of the oral cavity. From the above it isevident that the optimum compound to be administered to prevent and curemicrobial affections of the oral cavity must have an antibacterial andanti-adhesive activity like the protein components of saliva.

Currently, periodontitis and caries are treated by mechanically removingthe dental plaque and the damaged tissues and by surgically correctingand reconstructing the damaged structures. Furthermore, restoration ofoptimum oral hygiene conditions by using mouthwashes is very importantin preventing and curing these affections. However, treatment withmouthwashes based on disinfectant substances is not the ideal adjuvanttreatment in preventing and curing these disorders. These substances(e.g. chlorhexidine) in fact have a systemic toxicity and a markedincidence of side effects that limit their use; furthermore, thesesubstances do not prevent adhesion of microorganisms to dental surfaces.

DISCLOSURE OF THE INVENTION

The present invention relates to a protein formula or preparation forthe prevention and therapy of periodontitis and diseases of the teethand of the tissues of the oral cavity, characterized in that itcomprises a mixture of proteins comprising at least one transferrin in amixture with at least one additional protein selected from the groupcomprising albumins, lysozymes, protease inhibitors, and mixturesthereof; with the proviso that when said mixture does not contain eitheran albumin or a protease inhibitor, the transferrin and lysozymecomponents of the mixture are the only antibacterial, anti-adhesive oranti-invasive agents in said preparation.

It has been observed that the preparation according to the inventionperforms a synergistic activity and an antibacterial, anti-adhesive,anti-invasive and anti-protease activity that is not only higher thanthat of the individual proteins forming the mixture but also higher thanthat of salivary proteins.

Since at the present time there is no animal species that is considereduniversally valid for the testing of substances for preventing andcuring periodontitis, the following examples were applied to "in vitro"models that are generally accepted for the testing of adjuvants inpreventing and curing periodontitis and other bacterial disorders of theoral cavity.

Anti-adhesive activity is demonstrated by the reduced binding of themicroorganisms to a substance (hydroxyapatite) that simulates the dentalsurface; antibacterial activity is demonstrated by a bacteriostaticand/or bactericidal effect on specific bacterial genera; anti-proteaseactivity is verified by a higher anti-adhesive and antibacterialactivity of the complete protein formula with respect to the formulawithout inhibitors, which is subject to the aggression on the part ofmicrobial proteases. The anti-adhesive and antibacterial effectivenessof the protein formula according to the present invention is in facthigher than that obtained with its individual components, thus showing asynergistic activity due to the simultaneous presence of the differentproteins that characterize the formula described herein.

It has been observed that the quantitative ratios of the proteincompounds in the preparations according to the invention can vary withinwide limits, since these compounds already start having a synergisticeffectiveness at low concentrations in the mixture.

In particular, in binary mixtures of transferrin with an albumin or alysozyme, each one of the protein compounds can be present at aconcentration of 5 to 95% by weight with respect to the protein mixture.For example, preparations according to the invention can contain 10-40%transferrin and 90-60% albumin, or 90-60% transferrin and 10-40%lysozyme; the amounts may also be determined by economic factors, forexample by the cost of the proteins used.

In binary mixtures of transferrin with one or more protease inhibitors,said inhibitors can have concentrations of 1 to 20%, for example 2 to10%, by weight.

In preferred ternary mixtures of transferrin with albumin and lysozyme,total concentrations of transferrins and albumins can vary from 5 to 90%by weight, whereas lysozymes can be present at concentrations of 2 to90% by weight. For example, a ternary mixture can comprise 10-40%transferrin, 40-85% albumin and 2-20% lysozyme. When using a ternarymixture of transferrin with protease inhibitors and with an additionalprotein selected from albumins and lysozymes, the protease inhibitorsare present at a concentration of 1 to 20% by weight, for example 2 to10%, whereas the concentrations of each one of the other proteincompounds can vary between 5 and 94%, for example from 20 to 75%, withrespect to the mixture.

WAYS OF CARRYING OUT THE INVENTION

A particularly preferred preparation according to the invention is amixture of all four types of protein compounds mentioned above, and thusa mixture of at least one transferrin at a concentration of 5 to 92%, ofat least one lysozyme at a concentration of 2 to 80%, of at least onealbumin at a concentration of 5 to 92%, and of at least one proteaseinhibitor at a concentration of 1 to 20%, these concentrations beinggiven by weight with respect to the protein mixture. For example, such aquaternary mixture can comprise one or more transferrins at an overallconcentration of 10-50%, for example of 15-25%, one or more albumins atan overall concentration of 40-84%, for example of 60-75%, one or morelysozymes at an overall concentration of 5-30%, for example of 5-10%,and one or more protease inhibitors at a total concentration of 1-10%,for example of 2-5%, by weight.

By way of example, a preparation according to the invention cancomprises 75% albumin, 17% transferrin, 5% lysozyme and 3% proteaseinhibitors.

When the preparation according to the invention comprises more than onecompound of the class of transferrins, lysozymes, albumins or proteaseinhibitors, their combined amount totals the concentrations given abovefor each class of protein compounds.

The examples reported above show that the formula according to thepresent invention can be considered optimum, with respect to treatmentscurrently in use, for the prevention and cure of periodontitis and otherbacterial disorders of the oral cavity. Furthermore, the extremely lowtoxicity of the proteins contained in the formula according to thepresent invention is well-known, since these are extracted "natural"substances (but also chemically synthesizable) which are very similar,in their amino acid composition and structural configuration, to thehomologous proteins that are normally present in the human and animalbody. This is an additional advantage of the formula according to thepresent invention with respect to the chemicals currently used inperiodontitis treatment.

The proteins that can be used in the formula according to the presentinvention can therefore be extracts or can be obtained byrecombinant-DNA technology or also by chemical synthesis.

The term albumin designates, in the present invention, a glycoprotein ordeglycosylated protein having characteristics similar to those of humanalbumin; the formula according to the present invention can be producedwith any one of the proteins of various origin that are commonly termed"albumins" and are commercially available: serum albumin and lactalbuminfrom humans, bovines, cats, dogs, rabbits, horses, sheep, goats, serumalbumin and ovalbumin from turkeys, chickens, pigeons, etc.

The term transferrin designates a glycoprotein or a correspondingdeglycosylated protein capable of chelating two Fe(III) atoms permolecule, having structural and functional characteristics similar tothose of human lactoferrin, characterized by a known antibacterialactivity; the formula according to the present invention can be producedwith any one of the proteins of various origin commonly termed"transferrins" that are commercially available: serum transferrin andlactoferrin from humans, bovines, cats, dogs, rabbits, horses, sheep,goats, serum transferrin and ovotransferrin from turkeys, chickens,pigeons etc.

The term lysozyme designates a protein having structural and functionalcharacteristics similar to those of human lysozyme, which ischaracterized by a known enzymatic bactericidal activity againstGram-positive bacteria; the formula according to the present inventioncan be produced with any one of the proteins of various origin commonlytermed "lysozyme" that are commercially available: lysozyme from humans,bovines, horses, turkeys, chickens, etc.

The term protease inhibitors designates a class of proteins havingcharacteristics similar to those of human trypsin and chymotrypsininhibitors, characterized by the ability to inhibit the proteaseactivity of trypsin and chymotrypsin; the formula according to thepresent invention can be obtained with any one of the proteins ofvarious origin commonly termed "trypsin and chymotrypsin inhibitors"that are commercially available: trypsin and chymotrypsin inhibitorsfrom bovines, soybean seeds, chickens, turkeys, etc.

There are many proteins pertaining to the classes of albumins,transferring, lyzozymes and protease inhibitors, which are available onthe market and which can be used in the preparation of this invention.Illustrative of such commercial proteins are for example those sold bySIGMA Chemical Co., St. Louis, U.S.A.

The protein formula according to the present invention can be obtainedand stored in liquid form, as solutions at a concentration of 0.1 to10%, e.g. 1 to 5%, weight/volume (g/ml) of the protein mixture insolvents acceptable for pharmaceutical use, in particular water orhydro-alcoholic solvents such as water-ethanol mixtures, or in solidform (lyophilized, dried, frozen) and in the other commonly known formsof storage: for example immobilized or adsorbed on an inert supportcommonly used in the pharmaceutical field.

The protein preparation according to the invention can thus be used inthe liquid form, as a rinse or mouthwash, or in solid form, such as inpowder or granular form to be dissolved in water for preparing amouthwash just before use, with the concentrations specified above.Alternatively) it is possible to incorporate the protein mixture of theinvention in a toothpaste or in a formulation to be chewed, such aschewing gum, tablets, pastilles, lozenges, etc. in a concentration of 1to 60% by weight of the total formulation.

In its form ready-for-use the preparation according to the invention canalso comprise further conventional antibacterial and antiplaquecompounds as well as carriers, fillers, flavouring agents,preservatives, surfactants, colorants and other adjuvants selected fromthose conventionally used for the various liquid or solid formpreparations for oral topical use.

Thus, formulations for oral use according to the invention can alsocomprise further anti-bacterial and antiplaque compounds, such asquaternary ammonium compounds with one long chain alkyl on the nitrogenatom, alkali metal pyrophosphates and orthophosphates halogenatedbisphenols and halogenated diphenyl ethers, sodium benzoate, sodiumsalicylate, etc.

Liquid rinse formulations according to the invention can furthercomprise humectants, e.g. glycerin, sorbitol, xylitol, propylene glycol,etc., flavours, e.g. oil of spearmint, peppermint or cinnamon, menthol,methyl salicylate, etc., sweetening agents, e.g. aspartame, saccharin,dextrose, cyclamate, wintergreen, etc., thickening agents, e.g. xanthangum, carrageenin, carboxy methyl cellulose, etc., detergent builders,e.g. sodium carbonate, bicarbonate, sulfate or borate, etc., surfactantseither of anionic type, such as salts of higher fatty acid monoglyceridemonosulfates, higher alkyl sulfates, alkyl aryl sulfonates, higher alkylsulfoacetates, etc., or of non-ionic type, such as block polymers ofpolyoxyethylene and polyoxypropylene, polyethylene oxide condensates ofalkyl phenols, products of condensation of ethylene oxide with propyleneoxide and ethylene diamine, long chain tertiary amine oxides, long chaindialkyl sulphoxides, etc.

The toothpaste formulations and the formulations to be chewed by theuser will comprise the respective conventional base material andconventional adjuvant such as flavouring, sweetening and coloringagents, humectants, etc., as those mentioned above, and thickening andgelling agents such as thickening silica, natural or synthetic gums,e.g. tragacanth gum , guar gum, hydroxyethyl- and carboxymethylcellulose, polyvinyl pyrrolidone, starch, etc.

The protein preparation according to the invention, in either liquid orsolid form, should be used for purposes of prevention of periodontitisat least once, preferably twice a day. For purposes of treatment ofperiodontitis or other bacterial disorders the frequency of use can beincreased to 3 to 4 times a day.

A protein mixture according to the present invention was prepared,called hereinafter FORMULA, comprising 75% of bovine serum albumin, 17%of lactoferrin, 5% of lysozyme from chicken egg and 3% of trypsininhibitor from chicken egg, and was tested for antibacterial activity byplacing both the FORMULA itself and individual proteins composing it incontact with two microorganisms chosen as representatives of themicrobial flora that is commonly indicated as being responsible for thepathogenesis of periodontitis and of other tooth diseases. Antibacterialactivity was also measured by using the free proteins in solution orbound to hydroxyapatite, a substance used as a model of the dentalsurface.

The protein FORMULA of above was also tested for anti-adhesive activityby measuring its ability to prevent the binding to hydroxyapatite, asubstance used as a model of the dental surface, of two microorganismschosen as representatives of the microbial flora commonly consideredresponsible for the pathogenesis of periodontitis and of other toothdiseases.

The following microorganisms, chosen as representatives of the microbialflora commonly considered responsible for the pathogenesis ofperiodontitis and of other tooth diseases, were used:

Streptococcus mutans ATCC 6715-13 grown in Todd Hewitt Broth (THB-BBL)at 37° C.

Porphyromonas gingivalis grown at 37 ° C. in brain-heart infusion broth(BHI-BBL) or other known culture media, with or without the addition ofhaemin (10 μg/ml) and vitamin K (1 μg/ml), using an anaerobe hood(FORMA-Scientific, Marietta, Ohio).

EXAMPLE 1 Antibacterial Activity

The antibacterial activity of the following protein preparations wasassessed: lactoferrin, bovine serum albumin, salivary proteins, and theFORMULA of the present invention. The antibacterial activity of theabove-mentioned protein preparations was assessed by using them in freeform or adsorbed on hydroxyapatite. Efficiency was evaluated bymicrobial count in media suitable for the development of the teststrains, with or without the addition of the described substances. Thedegree of antibacterial activity of the individual proteins, as well asof the formula itself, can vary according to the biochemical and cultureconditions of the media in which testing occurs. Bacteriostatic activitycan in fact be detected in culture media during microbialmultiplication, whereas bactericidal activity can be already detected inthe first hour of contact between bacteria and proteins in salinesolution (PBS).

In a first test, 10⁵ cells/ml of Streptococcus mutans were added to thedescribed culture medium, which contained respectively 1 mg/ml oflactoferrin (Lf) or 1 mg/ml of bovine serum albumin (BSA) or 1 mg/ml ofsalivary proteins (PS) or 6 mg/ml of the FORMULA according to thepresent invention, which contained 1 mg/ml of the respectivetransferrin.

After 6 hours of incubation under agitation at 37° C., thecolony-forming units were counted (see Table 1a).

In a second test, 10⁵ cells/ml of Streptococcus mutans were added to thedescribed culture medium, which contained respectively 1 mg/ml oflactoferrin (Lf) or 1 mg/ml of bovine serum albumin (BSA) or 1 mg/ml ofsalivary proteins (PS) or 6 mg/ml of the FORMULA according to thepresent invention, which contained 1 mg/ml of the respectivetransferrin. In this second experiment, the above-mentioned proteins hadbeen kept in contact beforehand with 10 mg/ml of hydroxyapatite for 30minutes at 37° C., in order to allow adsorption of the proteins on thehydroxyapatite.

After 6 hours of incubation under agitation at 37° C., thecolony-forming units were counted (see Table 1a). The control, CTRL,consisted of the THB-BBL culture broth mentioned hereinabove, withoutprotein additions.

In a third test, 10⁵ cells/ml of Porphyromonas gingivalis were addedunder anaerobic conditions to the described culture media, whichcontained respectively 1 mg/ml of lactoferrin (Lf) or 1 mg/ml of bovineserum albumin (BSA) or 1 mg/ml of salivary proteins (PS) or 6 mg/ml ofthe FORMULA according to the present invention, which contained 1 mg/mlas transferrin.

After 48 hours of incubation under anaerobic conditions at 37° C., thecolony-forming units were counted (see Table 1b).

In a fourth test, 10⁵ cells/ml of Porphyromonas gingivalis were addedunder anaerobic conditions to the described culture media, whichcontained respectively 1 mg/ml of lactoferrin (Lf) or 1 mg/ml of bovineserum albumin (BSA) or 1 mg/ml of salivary proteins (PS) or 6 mg/ml ofthe FORMULA according to the present invention, which contained 1 mg/mlas transferrin. In this fourth example, the above-mentioned proteins hadbeen kept in contact beforehand with 10 mg/ml of hydroxyapatite for 30minutes at 37° C., in order to allow adsorption of the proteins on thehydroxyapatite.

After 48 hours of incubation under anaerobic conditions at 37° C., thecolony-forming units were counted (see Table 1b). The CTRL consisted ofthe BHI-BBL culture broth mentioned hereinabove.

                  TABLE 1a                                                        ______________________________________                                        Antibacterial activity after 6 hours of                                       incubation at 37° C. under agitation. The inoculum                     consisted of 10 cells/ml of S. mutans.                                                   CTRL    Lf      BSA   PS    FORMULA                                ______________________________________                                        Free proteins                                                                            5 × 10.sup.8                                                                    1 × 10.sup.6                                                                    5 × 10.sup.8                                                                  5 × 10.sup.6                                                                  5 × 10.sup.4                     Streptococcus                                                                 mutans                                                                        Proteins adsorbed                                                                        5 × 10.sup.8                                                                    5 × 10.sup.5                                                                    5 × 10.sup.8                                                                  2 × 10.sup.6                                                                  2 × 10.sup.4                     on hydroxyapatite                                                             ______________________________________                                    

                  TABLE 1b                                                        ______________________________________                                        Antibacterial activity after 48 hours of                                      incubation at 37° C. under anaerobic conditions. The inoculum          consisted of 10.sup.5  cells/ml of Porphyromonas gingivalis.                             CTRL    Lf      BSA   PS    FORMULA                                ______________________________________                                        Free proteins                                                                            5 × 10.sup.5                                                                    1 × 10.sup.5                                                                    5 × 10.sup.5                                                                  1 × 10.sup.4                                                                  1 × 10.sup.4                     Porphyromonas                                                                 gingivalis                                                                    Proteins adsorbed                                                                        5 × 10.sup.5                                                                    1 × 10.sup.5                                                                    5 × 10.sup.5                                                                  2 × 10.sup.4                                                                  5 × 10.sup.3                     on hydroyapatite                                                              ______________________________________                                    

Naturally, the higher the antibacterial activity, the lower the numberof microorganisms detected after incubation. The above-reported exampleclearly shows that the formula according to the present inventionexhibits the maximum antibacterial activity both against Streptococcusmutans and against Porphyromonas gingivalis.

EXAMPLE 2 Anti-adhesive Activity

The anti-adhesive activity of the following proteins was assessed:lactoferrin, bovine serum albumin, salivary proteins, the FORMULAaccording to the present invention.

Columns with a diameter of 0.8 cm for low-pressure liquidchromatography, containing 1 g of hydroxyapatite in a phosphate buffer(PBS), were prepared.

After percolation of the protein solutions containing respectively 100mg/10 ml of lactoferrin (Lf) or 100 mg/10 ml of bovine serum albumin(BSA) or 100 mg/10 ml of salivary proteins (PS) or 100 mg/10 ml of theFORMULA according to the present invention, the proteins that had notadhered were removed by washing with 100 ml of PBS. Appropriate dosagesof the proteins in the eluate of the wash showed that 80% of thepercolated proteins had remained adsorbed on the gram of hydroxyapatitecontained in the chromatography column.

The chromatography columns were subsequently inoculated under aerobic oranaerobic conditions with a suspension containing 10⁸ cells of thestrains of Streptococcus mutans or Porphyromonas gingivalis in aphysiological solution. Washing with PBS (1000 ml) eluted themicroorganisms that had not adhered to the hydroxyapatite.

A second wash with 100 ml of NaCl 1M allowed elution of themicroorganisms adhered to the hydroxyapatite (Table 2). The controls inTable 2 consisted of the columns prepared as disclosed, i.e. ofhydroxyapatite without addition of proteins.

                  TABLE 2                                                         ______________________________________                                        Anti-adhesive activity                                                                   CTRL    Lf      BSA   PS    FORMULA                                ______________________________________                                        Streptococcus                                                                 mutans                                                                        PBS wash   7 × 10.sup.2                                                                    9 × 10.sup.7                                                                    9 × 10.sup.7                                                                  5 × 10.sup.7                                                                  1 × 10.sup.8                     1M NaCl wash                                                                             9 × 10.sup.7                                                                    1 × 10.sup.5                                                                    6 × 10.sup.6                                                                  4 × 10.sup.7                                                                  1 × 10.sup.3                     Porphyromonas                                                                 gingivalis                                                                    PBS wash   1 × 10.sup.2                                                                    8 × 10.sup.7                                                                    8 × 10.sup.7                                                                  4 × 10.sup.7                                                                  9 × 10.sup.7                     1M NaCl wash                                                                             1 × 10.sup.8                                                                    7 × 10.sup.6                                                                    8 × 10.sup.6                                                                  6 × 10.sup.7                                                                  1 × 10.sup.4                     ______________________________________                                    

The higher the anti-adhesive ability of a substance, the lower thenumber of microorganisms eluted with 1M NaCl, since the microorganismsthat had not adhered were washed away beforehand by PBS. The aboveexample clearly shows that the formula according to the presentinvention exhibits the highest anti-adhesive ability both againstStreptococcus mutans and against Porphyromonas gingivalis.

EXAMPLE 3

The antibacterial activity of various preparations according to theinvention was evaluated with respect to individual components and to acontrol sample containing no protein compounds, tested according to theabove-described experimental conditions (first and third tests ofExample 1).

The proteins were added at the following concentrations: transferrin(TRF) 1 mg/ml; lysozyme (LYS) 0.3 mg/ml; protease inhibitors (INHIB)0.20 mg/ml; albumin (ALB) 4.5 mg/ml. The control (CTRL) consisted of theculture broths used in said first and third tests of example 1, to whichno protein was added.

    ______________________________________                                        PROTEINS           S. mutans                                                                              P. gingivalis                                     ______________________________________                                        CTRL               5 × 10.sup.8                                                                     5 × 10.sup.5                                TRF                1 × 10.sup.6                                                                     1 × 10.sup.5                                TRF + LYS          1 × 10.sup.5                                                                     5 × 10.sup.4                                TRF + LYS + ALB    1 × 10.sup.5                                                                     5 × 10.sup.4                                TRF + LYS + INHIB  8 × 10.sup.4                                                                     2 × 10.sup.4                                TRF + LYS + ALB + INHIB                                                                          5 × 10.sup.4                                                                     1 × 10.sup.4                                ______________________________________                                    

The following examples show various ready-to-use forms of thepreparations according to the invention.

EXAMPLE 4 Mouthwash

Composition per 100 ml

active ingredients:

Albumin, serum albumin from bovine serum, SIGMA A6793, 650 mg

Transferrin, ovotransferrin from chicken egg, SIGMA C1130, 200 mg

Lysozyme, from chicken egg, SIGMA L2879, 100 mg

Trypsin inhibitor, from soy bean, SIGMA T9128, 50 mg

carriers, preservatives and flavouring agents:

Sodium chloride 1 g

Sodium bicarbonate 100 mg

Methyl-p-hydroxybenzoate 100 mg

Peppermint oil 50 mg

Purified water to 100 ml

use:

Two rinses a day, one in the morning, one in the evening.

EXAMPLE 5 Toothpaste

Composition per 100 g

active ingredients:

Albumin, serum albumin from bovine serum, SIGMA A6793, 1.5 g

Transferrin, lactoferrin from bovine milk, SIGMA L9507, 0.5 g

Lysozyme, from chicken egg, SIGMA L2879, 100 mg

Trypsin inhibitor, from chicken egg, SIGMA T9253, 50 mg

carriers, preservatives and flavouring agents:

Precipitated silica 15 g

Sodium benzoate 4 g

Sodium lauryl sulfate 2 g

Calcium phosphate 1.2 g

Sodium carrageenin 1.1 g

Titanium dioxide 1 g

Sodium mono-fluoro phosphate 0.3 g

Methyl-p-hydroxybenzoate 0.1 g

Peppermint oil 50 mg

Purified water to 100 g

use:

Use as a normal toothpaste twice a day, in the morning and

evening.

EXAMPLE 6 Envelope Packs

Composition for 1 envelope

active ingredients:

Albumin, lactalbumin from bovine milk, SIGMA L5385, 150 mg

Transferrin, ovotransferrin from chicken egg, SIGMA C1130, 35 mg

Lysozyme, from chicken egg, SIGMA L2879, 10 mg

Trypsin inhibitor, from chicken egg, SIGMA T9253, 5 mg

carriers, preservatives and flavouring agents:

Sodium chloride 20 mg

Sodium bicarbonate 10 mg

Methyl-p-hydroxybenzoate 20 mg

Peppermint oil 5 mg

The ingredients are reduced to finely divided powder and thoroughlymixed on conventional grinding and mixing equipment.

use:

The content of an envelope is dissolved in 20 ml water for two rinseuses, one in the morning and one in the evening.

EXAMPLE 7 Chewing Gum

Composition of one piece of gum:

active ingredients:

Albumin, ovoalbumin from chicken egg, SIGMA A5253, 150 mg

Transferrin, serum transferrin from bovine serum, SIGMA T5761, 35 mg

Lysozyme, from chicken egg, SIGMA L2879, 10 mg

Trypsin inhibitor, from soy bean, SIGMA T9128, 5 mg

carriers, preservatives and flavouring agents:

Gum base (Paloya TX) 400 mg

Glucose 100 mg

Glycerol 10 mg

Sodium bicarbonate 10 mg

Methyl-p-hydroxybenzoate 20 mg

Peppermint oil 5 mg

use:

One piece of chewing gum after the main meals.

We claim:
 1. Topical preparation for use in the prevention and therapyof diseases of the teeth and of the oral cavity caused by bacteria oradhesion of pathogenic species, characterized in that it comprises amixture of proteins comprising at least one transferrin at aconcentration of 5 to 95% by weight; at least one albumin at aconcentration of 5 to 95% by weight; and at least one lysozyme at aconcentration of 2 to 90% by weight.
 2. Preparation according to claim1, comprising a mixture of said at least one transferrin with at leastone protease inhibitor and with at least one protein selected fromlysozymes and albumins, wherein said inhibitor is present at aconcentration of 1 to 20% by weight, said at least one transferrin andat least one lysozyme or at least one albumin being each present at aconcentration of 5 to 94% by weight.
 3. Preparation according to claim1, wherein said protein mixture comprises 5 to 92% of said at least onetransferrin, 5 to 92% of said at least one albumin, 2 to 80% of said atleast one lysozyme, and 1 to 20% of said at least one proteaseinhibitor, said percentages being expressed by weight with respect tothe weight of the protein mixture.
 4. Preparation according to claim 3,wherein said protein mixture comprises 10 to 50% of said at least onetransferrin, 40 to 84% of said at least one albumin, 5 to 30% of said atleast one lysozyme and 1 to 10% of said at least one protease inhibitor.5. Preparation according to claim 3, wherein said protein mixturecomprises 15 to 25% of said at least one transferrin, 60 to 75% of saidat least one albumin, 5 to 10% of said at least one lysozyme and 2 to 5%of said at least one protease inhibitor.
 6. Preparation according toclaim 1, wherein said proteins are selected from the group consisting ofsaid proteins of natural origin, comprising proteins of human, animaland plant origin, proteins obtained by recombinant-DNA technology andchemically synthesized proteins.
 7. Preparation according to claim 1, ina form selected from the group comprising a liquid preparation, a pastypreparation, a solid preparation and a preparation immobilized oradsorbed on an inert support.
 8. Preparation according to claim 7 in theform of a mouthwash comprising from 0.1 to 10% w/v of said proteinmixture in a solvent selected from water and ethanol-water mixtures. 9.Preparation according to claim 8 further comprising one or moreadjuvants selected from humectants, sweeteners, flavouring agents,thickening agents, detergent builders, surfactants and antibacterial andantiplaque agents, acceptable for oral topical use.
 10. Preparationaccording to claim 7 in a form selected from toothpaste and formulationsto be chewed, comprising from 1 to 60% by weight of said protein mixtureincorporated in a conventional base for a toothpaste or a formulation tobe chewed.
 11. Preparation according to claim 10 further comprising oneor more adjuvants selected from thickening agents, humectants,sweeteners, flavoring agents, and antibacterial and antiplaque agentsacceptable for oral topical use.
 12. A method of using the preparationaccording to claim 1 for preventing and treating diseases of the teethand of the oral cavity of a mammal caused by bacteria or adhesion ofpathogenic species which comprises topically administering to saidmammal an amount of the preparation according to claim 1 sufficient forpreventing and treating diseases of the teeth and of the oral cavitycaused by bacteria or adhesion of pathogenic species.
 13. Preparationaccording to claim 1, further including at least one protease inhibitor.